Mycobacterium tuberculosis is a highly successful human pathogen that has evolved to survive within the host. Our long-term goals are to delineate the mechanisms by which M. tuberculosis interferes with the microbicidal functions of macrophages and evades host immune responses. Understanding the molecular mechanisms underlying M. tuberculosis-}r\osi interactions is important for designing therapeutic interventions and vaccine strategies. We have identified Rv2224c, a predicted protease, as being critical for survival in macrophages and in vivo. We hypothesize that Rv2224c is an important virulence factor that proteolytically cleaves M. tuberculosis substrates and modulates the host immune response. Our preliminary studies indicate that Rv2224c is exported to the cell envelope of mycobacteria and secreted extracellularly. A mutant disrupted in the protease is impaired for growth in macrophages and in vivo. Mice infected with a protease mutant show markedly reduced lung pathology and survive longer than wildype-infected mice. In this proposal will study the role of the protease in host-pathogen interactions. The specific aims are: 1. Molecular and biochemical characterization of Rv2224c and its potential substrate(s) in M. tuberculosis. We will study the localization of Rv2224c in mycobacteria and perform structure-function analysis of Rv2224c. We will investigate the biological activity and specificity of the protease by studying a candidate physiologic substrate. 2. Study the interaction of the protease with the host immune response in macrophages and in vivo. We will examine the nature of the immune response to Rv2224c mutant, specifically determine the role of T and B cells in immunity and perform structure-function analysis of protease interaction with the host in macrophages and in vivo. 3. Dissect the modulation of macrophage function by the protease. We will ask whether infection with the mutant elicits altered macrophage responses and test whether the Rv2224c::tn mutant is hyper-susceptible to IFNy-mediated immune responses. Finally, we will study whether Rv2224 directly interacts with host cell components using proteinprotein interaction studies.